The research project proposed is aimed at the description, characterization and correlation of events involved in excitation-contraction coupling in twitch muscle fibers. Experiments are proposed for studying events occurring in tubular system membranes, triad and sacroplamsic reticulum membranes in addition to calcium concentration changes in thh sarcoplasm. The membrane potential changes of the tubular system and the sarcoplasmic reticulum will be monitored by voltage-sensing-dyes (fluorescence and absorption). Triadic function will be studied in correlation to charge movements. Calcium concentration changes will be monitored by calcium-sensing-dyes. Modifiers of excitation-contraction coupling will be tested to assess the role of each of the above processes in the physiology of muscle contractions. We will use a cut fiber preparation and double vaseline gap voltage clamp technique allowing modification of the internal medium of the muscle fiber, control of the surface membrane potential and measurement of optical signals. Our experiments are expected to provide new information on 1) the steps involved in excitation-contraction coupling, and 2) the links between consecutive steps. These studies are expected to be of importance in the physiology of striated muscle, including heart muscle.